Fludarabine Phosphate Injection

» Fludarabine Phosphate Injection is a sterile solution of Fludarabine Phosphate in Water for Injection. It contains not less than 95.0 percent and not more than 105.0 percent of the labeled amount of fludarabine phosphate (C10H13FN5O7P).

[Caution—Fludarabine Phosphate is potentially cytotoxic. Great care should be taken to prevent inhaling particles and exposing the skin to it. ]

Packaging and storage— Preserve in well-closed containers, preferably of Type I glass, protected from light. Store in a refrigerator.

USP Reference standards

—
USP Fludarabine Phosphate RS.

Identification—

A: Ultraviolet Absorption

197U

—

Solution: 27 µg per mL.

Medium: 0.1 M hydrochloric acid.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Bacterial endotoxins

— It contains not more than 7.7 USP Endotoxin Units per mg of fludarabine phosphate.

Sterility

— It meets the requirements when tested as directed for Membrane Filtration under Test for Sterility of the Product to be Examined.

791

: between 6.0 and 7.1.

Particulate matter

788

: meets the requirements.

Related compounds— [note—Fludarabine Phosphate Injection should comply with both Test 1 and Test 2.]

test 1—

Buffer solution and Mobile phase— Prepare as directed in the Assay.

Standard solution— Quantitatively transfer 1.0 mL of the Standard preparation, prepared as directed in the Assay, into a 100-mL volumetric flask. Dilute with Buffer solution to volume, and mix to obtain a solution having a known concentration of 0.001 mg of fludarabine phosphate per mL.

Test solution— Use the Assay preparation.

Sensitivity solution— Quantitatively transfer 2.0 mL of the Standard solution into a 25-mL volumetric flask. Dilute with Buffer solution to volume, and mix to obtain a solution having a known concentration of 0.08 µg of fludarabine phosphate per mL.

Resolution solution— Add 3 drops of 2 N hydrochloric acid solution to 1.5 mL of the Standard solution. Mix well, and heat at 80

for 30 minutes.

Chromatographic system (see Chromatography

621

)— The liquid chromatograph is equipped with a 260-nm UV detector and a 4.6-mm × 25-cm column containing 5-µm packing L1. The flow rate is 1.0 mL per minute. Chromatograph the Sensitivity solution at 260 nm. The ratio of the fludarabine phosphate peak height to the noise height is not less than 10, the noise height being determined by a suitable procedure. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between the fludarabine phosphate peak and the 2-fluoroadenine peak (relative retention time about 1.3) is not less than 5. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 1.8 for the fludarabine phosphate peak, and the relative standard deviation for replicate injections is not more than 1%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, and record the chromatograms at 260 nm. Measure the response of the fludarabine phosphate peak for the Standard solution, and measure the responses of all the major peaks, excluding the fludarabine phosphate peak, for the Test solution. Calculate the percentage of each impurity present in the portion of Injection taken by the formula:

1000(C/F)(rU / rS)

in which C is the concentration, in mg per mL, of USP Fludarabine Phosphate RS in the Standard solution; F is a relative response factor (see Table 1 for values) and equal to 1.0 for any other impurities; rU is the peak response for each individual impurity in the Test solution; and rS is the peak response for fludarabine phosphate in the Standard solution. The limits of impurities are specified in Table 1.

Table 1

Impurity	Relative Response Factor (F)	Relative Retention Time	Limit (w/w, %)
Iso-ara-guanine monophosphate	0.28	0.45	0.4
2-Amino analog	0.45	0.54	0.2
Compound A	1.0	0.62	0.3
2-Fluoroadenine	1.0	1.3	0.2
Individual unspecified impurity	1.0	—	0.3
Total impurities	—	—	2.0

test 2—

Mobile phase, Standard solution, System suitability solution, Sensitivity check solution, Chromatographic system, Procedure, and Limits— Proceed as directed in Related compounds, Test A and Test B, under Fludarabine Phosphate for Injection.

Test solution— Quantitatively transfer 1.0 mL of Injection into a 25-mL volumetric flask. Dilute with Mobile phase to volume, and mix to obtain a solution having a concentration of about 1 mg of fludarabine phosphate per mL.

Other requirements— It meets the requirements under Injections

Assay—

Buffer solution— Dissolve 13.8 g of monobasic sodium phosphate monohydrate in 2000 mL of water (50 mM). Adjust with 1.0 N sodium hydroxide to a pH of 4.5 ± 0.2.

Mobile phase— Prepare a mixture of filtered and degassed Buffer solution and methanol (47:3).

Standard preparation— Dissolve an accurately weighed quantity of USP Fludarabine Phosphate RS in Buffer solution. Dilute quantitatively with Buffer solution to volume, and mix to obtain a solution having a known concentration of about 0.1 mg of fludarabine phosphate per mL.

Assay preparation— Quantitatively transfer 1.0 mL of Injection into a 250-mL volumetric flask. Dilute with Buffer solution to volume, and mix to obtain a solution having a concentration of about 0.1 mg of fludarabine phosphate per mL.

Chromatographic system (see Chromatography

621

)— The liquid chromatograph is equipped with a 260-nm UV detector and a 4.6-mm × 25-cm column containing 5-µm packing L1. The flow rate is 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not more than 1.8 for the fludarabine phosphate peak, and the relative standard deviation for replicate injections is not more than 1%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the response for the fludarabine phosphate peak. Calculate the quantity, in mg per mL, of C10H13FN5O7P in the portion of Injection taken by the formula:

250(C/V)(rU / rS)

in which C is the concentration, in mg per mL, of USP Fludarabine Phosphate RS in the Standard preparation; V is the volume, in mL, of Injection taken; and rU and rS are the peak responses for fludarabine phosphate in the Assay preparation and the Standard preparation, respectively.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Feiwen Mao, M.S. Scientist 1-301-816-8320	(MDOOD05) Monograph Development-Ophthalmics Oncologics and Dermatologicals
Reference Standards	Lili Wang, Technical Services Scientist 1-301-816-8129 RSTech@usp.org
85	Radhakrishna S Tirumalai, Ph.D. Senior Scientist 1-301-816-8339	(MSA05) Microbiology and Sterility Assurance
71	Radhakrishna S Tirumalai, Ph.D. Senior Scientist 1-301-816-8339	(MSA05) Microbiology and Sterility Assurance

USP32–NF27 Page 2388

Pharmacopeial Forum: Volume No. 33(2) Page 232

Chromatographic Column—

FLUDARABINE PHOSPHATE INJECTION

Chromatographic columns text is not derived from, and not part of, USP 32 or NF 27.